ABSTRACT De novo or acquired resistance to endocrine therapy is a key driver of relapse and tumor progression in patients with estrogen receptor (ER+) breast cancer. Downregulation of the ER? is a well-established yet poorly understand mechanism of endocrine resistance. Currently there are no approved or investigational therapies that can restore ER? expression after downregulation occurs. Our preclinical research was the first to demonstrate in luminal ER+ breast cancer a novel role of Aurora-A kinase (AURKA) in the induction of stemness reprogramming and expansion of a sub-population of CD44high/CD24low breast tumor initiating cells (BTICs). These BTICs have low ER? expression and are resistant to endocrine therapy. They are further characterized by stem cell-like phenotype that mediates tumor properties of invasion, self-renewal, drug resistance, and metastasis. We propose in ER+ breast cancer that activation of AUKRA induces expansion of BTICs during tumor progression that drives ER? downregulation and endocrine resistance. When BTICs are treated with the selective AURKA inhibitor, alisertib, the luminal phenotype, ER? expression and endocrine sensitivity are restored. Our preclinical data demonstrates synergy when alisertib is combined with fulvestrant in endocrine resistant models. We have recently determined the maximum tolerated dose of alisertib in combination with standard dose fulvestrant in a phase I clinical trial in advanced ER+ breast cancer. An excellent safety profile and impressive 6-month clinical benefit rate of 78% were observed. The objective of the proposed research is to explore in preclinical models and clinical biospecimens the molecular mechanisms by which AURKA promotes endocrine resistance through stemness reprogramming and downregulation of ER?. Our central hypothesis is that alisertib selectively targets BTICs and converts them to a CD44low/CD24high luminal phenotype with increased ER? expression and sensitivity to endocrine therapy. The following Aims will assess the central hypothesis: In Specific Aim 1 we will evaluate AURKA as a novel biomarker and therapeutic target in endocrine resistant, metastatic breast cancer. In Specific Aim 2 we will determine in vivo the mechanisms underlying AURKA-induced endocrine resistance and tumor progression. In Specific Aim 3 we will define the role of SMAD5/SOX2 transcriptional network in mediating AURKA-induced enrichment of CD44high/CD24low/ER?low BTICs. Dissecting the mechanisms by which AURKA induces stemness reprogramming is of high translational significance because AURKA inhibitors may represent a novel therapeutic approach to address de novo and acquired endocrine resistance due to ER? downregulation. The proposed research will establish the extent to which targeting AURKA has consequent benefit to delay or prevent tumor progression and improve the survival of patients with ER+ breast cancer. This research is directly relevant to the goals of the NIH because it will enhance our understanding of the mechanisms that drive endocrine resistance and breast cancer progression.